The present invention relates to the field of topical application of an enzyme-containing composition.
Enzymes are used in a number of consumer products, among which are detergents and cosmetics. A problem associated with enzyme-containing products is that enzymes may induce allergic sensitization upon inhalation of airborne dust or aerosols. For instance, after topical application of an enzyme-containing cream, enzyme-containing dust particles may be released from the site of application when the cream is dried.
One solution to prevent inhalation of enzyme-containing dust particles is to immobilize the enzyme before its actual application (see Japanese patent applications J57073098 and J61268611). However, the activity of an immobilized enzyme towards a solid substrate (the skin) is known to be quite low. For instance, immobilized proteases are not expected to interact with stratum corneum proteins.
It would therefore be desirable to apply a composition containing an enzyme in a non-immobilized form in such a way that release of dust particles from the site of application would not occur.
The present invention provides a convenient solution to the above problems by providing a method for topical application of an enzyme, wherein the release of enzyme-containing dust-particles from the site of application is prevented whereas enzyme activity is guaranteed.
The present invention discloses a method for topical application of an enzyme-containing composition, wherein an enzyme composition is applied which additionally comprises a film-forming agent. Upon topical application of said composition, the film-forming agent allows for the formation of a peelable film incorporating the enzyme, due to evaporation of the solvent(s) used. After evaporation of the solvent and after the enzyme has fulfilled its task, the film incorporating the enzyme is removed from the site of application. In this way, the release of enzyme-containing dust particles, which easily are formed upon drying of an enzyme-containing composition at the site of application, is prevented.
Specifically, the method of the invention comprises the steps of:
preparing a composition which comprises an enzyme and a film-forming agent,
topically applying said composition,
incubating said composition on the site of application for a sufficient time period to enable formation of a film incorporating the enzyme, and
removing said film incorporating the enzyme from the site of application.
In one embodiment of the invention, the composition comprising an enzyme and a film-forming agent is prepared by mixing two separate compositions, i.e. a first composition comprising the enzyme and a second composition comprising the film-forming agent. In this way, the enzyme is not detrimentally influenced by the presence of the dissolved film-forming agent or by the presence of any auxiliary agent.
A preferred option of this embodiment comprises the packaging of the said two individual compositions in a dual component dispensing system. Using a dual component dispensing system, simultaneous delivery of a composition comprising the enzyme and a composition comprising the film-forming agent is possible. Upon delivery, both individual compositions are mixed, either in situ or in the dispensing system. Mixing of both compositions results in a final composition comprising the enzyme and the film-forming agent in a form which is suitable for direct use.
The dispensing system to be used in this embodiment of the invention is not critical. Any system can be used which allows for the separate containment of the enzyme composition and the composition comprising the film-forming agent.
For instance, a dispensing system can be selected from the dual component dispensing systems which have been developed for the packaging and delivery of non-compatible chemical compounds, i.e. chemical compounds which react with each other when brought into contact. For instance, dual component dispensers are known from the field of adhesives.
Apart from adhesives, multicomponent dispensing systems have also been described for the formulation of incompatible compounds in toothpaste. Flexible two-compartment dentrifice tubes are described in U.S. patents U.S. Pat. No. 4,487,757 and U.S. Pat. No. 4,098,435. A two-compartment tube for the storage of a non-aqueous enzyme composition separate from the aqueous toothpaste composition has been described in FR 2,051,922.
In yet another but basically very simple approach, one pair of plastic pouches provides material for single use only. The outlets of the two pouches are close to each other and discharge of the contents can be effectuated by tearing open the end pieces of the pouch (German patent application DE 3 630 849).
Furthermore, German xe2x80x9cGebrauchsmusterxe2x80x9d DE, U, 93 07 726 mentions a two-chamber packaging unit in which the two separately packed components can be mixed by applying pressure on one of the chambers. Only after mixing is complete, the package is opened to release the freshly prepared mixture.
The commercially available container shown in FIG. 1 is also suitable. In this container, the enzyme composition, e.g. in the form of a lyophilized powder, is present in an insert placed on top of a vial. Pushing the cap downwards pierces the membrane separating the enzyme composition and the composition comprising the film-forming agent. Upon shaking, the enzyme composition and the film-forming composition are mixed and subsequently are applied.
Using the containers shown in FIGS. 2A and B, the same complete mixing approach is obtained. The container of FIG. 2A is a conventional tube comprising the composition with the film-forming agent, additionally containing a small sachet or ampulla containing the enzyme in a stabilized form. The container of FIG. 2B is a two-compartment sachet, wherein both compartments are separated by a breakable membrane. Just before use, the sachet or ampulla is opened or broken by bending or pressing in such a way that the stabilized enzyme is released. After thorough mixing of the enzyme with the composition comprising the film-forming agent, the tube or sachet is opened and its contents topically applied.
The enzyme to be used in the method of the invention will depend on the desired application. Suitable enzymes typically are found in the classes of oxidoreductases, transferases, hydrolases and/or isomerases. For example, the enzyme is a glucose oxidase, peroxidase, lipoxygenase, superoxide dismutase, tyrosinase, protease, phosphatase, phytase, glycosidase, glucanase, lysozyme, esterase, lipase, phospholipase, sulfatase, urease, transglutaminase or protein disulfide isomerase. It is also possible to apply a composition comprising a mixture of two or more enzymes.
A preferred enzyme for use in the method of the invention is a protease.
The way in which the enzyme is formulated is not critical for the invention. The enzyme may be formulated as a solid or as a liquid composition. A liquid composition may for instance be an aqueous composition wherein the enzyme is solubilized and, preferably, stabilized, or may be a suspension wherein a solid form of the enzyme is suspended in a liquid. Alternatively, the enzyme may be formulated as an oil-in-water emulsion or as a water-in-oil emulsion. In both emulsion types, the enzyme is present in the aqueous phase, preferably in a stabilized form. Furthermore, the enzyme can be presented as an essentially dry powder, optionally suspended in a non-aqueous liquid. The nature of the enzyme composition may thereby depend on, for instance, the desired application.
In a preferred embodiment of the invention, the enzyme composition is an aqueous composition wherein the enzyme is solubilized. To stably formulate an enzyme in an aqueous composition, the enzyme composition preferably comprises a sufficient amount of a water activity lowering agent. For instance, a polyol may be used for stabilization.
In that regard, the choice of the polyol which is used to stabilize an aqueous enzyme composition is not critical for the invention. Any polyol which is known to the skilled person to effectively stabilize an enzyme in an aqueous solution can be used. Polyols that are particularly useful are polyols selected from the group of glycerol, sorbitol, propylene glycol, maltodextrins, or a sugar such as sucrose, lactose, glucose or trehalose. For topical applications, one should consider a polyol which is acceptable for topical use, i.e. glycerol, polyethylene glycol, butylene glycol, propylene glycol, trehalose or sorbitol.
The polyol is used in a high concentration, i.e. a concentration which results in a sufficiently low water activity in the enzyme composition to adequately stabilize the enzyme. It is known in the art that these concentrations may somewhat vary with the polyol used. Preferably, the polyol is used in a concentration of 20-100%, more preferably in a concentration of 30-90%, even more preferably in a concentration of 40-90%, even more preferably in a concentration of 50-90%, most preferably in a concentration of 60-80%.
Such a stable aqueous composition can also be used to produce, when combined with a suitable emulsifier, either a water-in-oil emulsion (see WO 95/28092) or an oil-in-water emulsion.
In another preferred embodiment of the invention, the enzyme composition is an aqueous composition which is a suspension of the enzyme in a precipitated form. For instance, the enzyme is precipitated by the addition of a high concentration of a salt, such as ammonium sulphate.
The film-forming agent can be any agent which allows for both the formation of a peelable mask (peelable film) upon evaporation of solvent occurring after topical application of the composition comprising the enzyme and the film-forming agent as well as the release of 90% or more of the enzyme being incorporated in said peelable film.
For instance, the film-forming agent can be a soluble or dispersible colloid, including a broad range of polymeric compounds like polyvinyl alcohol, acrylic (co)polymers, polyurethane, latex, polyvinylpyrrolidone, hydrocolloids like gum karaya or alginate, and the like. The choice of the film-forming agent to be used may among others be dependent on the enzyme which is intended to be topically applied. Said choice further may be dependent on the desired solubility, tacticity, plasticity, rheology, and drying time. Apart from these aspects, shelf stability may also taken into account.
A preferred film-forming agent to be used in the composition according to the invention is polyvinyl alcohol (PVA). It is generally known to the skilled person that the properties of a PVA film will mainly depend on the type of PVA which is used for its preparation. The properties of the various types of PVA are mainly governed by the molecular weight (degree of polymerization) and the remaining content of acetyl groups (see for instance the Hoechst High Chem brochure on Mowiol PVA, as can be obtained from Clariant Benelux, Amsterdam, the Netherlands).
The specific type of PVA which may be selected is also dependent on the general aspects taken into account for the choice of a specific film-forming agent.
If desired, mixtures of different PVA types may be applied to obtain a film having optimal characteristics.
For instance, a suitable PVA for use in combination with an aqueous enzyme-containing composition is a PVA which gives a satisfactory viscosity in the presence of a substantial amount of water. The viscosity of a PVA formulation thereby typically will depend on the type of PVA as well as the concentration of the PVA.
In addition to a film-forming agent, compounds may be present in the composition according to the invention which may influence the properties of the composition and/or the resulting film.
For instance, to obtain a very viscous solution, a suitable viscosifier, such as xanthan or hydroxyethylcellulose may be added.
A high amount of water in the composition according to the invention allows the enzyme to act on the skin for a relatively long time period. However, a relatively short drying time may be more convenient for the user. To shorten the drying time of the PVA solution, it may be an option to lower the water content of the formulation. For instance, the aqueous phase of the composition may further contain a solvent to hasten evaporation, like ethanol.
To lower the water content of the formulation even further, a low viscous PVA grade, for instance Mowiol 3-83 or 4-88 (Clariant), can be used, since a low viscous PVA grade may be applied in a relatively high concentration. With these low viscous PVA grades solutions up to 35% (w/w) in water yield an acceptable spreadability. A withcoming advantage of such relatively high PVA concentrations is an increased tear strength of the film formed upon drying.
To produce a film with a varying degree of plasticity, a plasticizing agent may be used, like glycerol, sorbitol, ethylene glycol.
To minimize tacticity, a so-called adhesion reducer may be added. Examples of such compounds are silicone oil or certain waxes. To increase tacticity, certain cosmetically acceptable compounds like for example honey may be added.
To improve spreadability, drying behaviour and removability, the composition of the invention may comprise a surfactant functioning as a wetting agent, e.g. a non-ionic surfactant, like Tween 20, and/or a betaine.
The PVA is applied in the formulation in a concentration which may vary from 0.1-40%. A high viscosity PVA grade, like Mowiol 40-88 (Clariant), typically is applied in the lower concentration ranges, e.g. from 0.1-20%, preferably from 1-10%, whereas a low viscosity PVA grade, like Mowiol 3-83 or 4-88, typically is applied in the higher ranges, e.g. from 20-40%, preferably from 25-30%.
The composition according to the invention may include other active ingredients next to an enzyme and a film-forming agent.
For instance, the composition according to the invention may include a substrate for the enzyme which is present in the composition. Examples are linoleic or linolenic acid as a substrate for lipoxygenase; casein as a substrate for transglutaminase; hydrogen peroxide as a substrate for peroxidase; vitamin precursors as a substrate for esterases/lipases.
In addition, the composition according to the invention may include an active ingredient which acts synergistically with the enzyme of interest.
It is up to the choice of the skilled person whether the additional active ingredient is present in composition comprising the film-forming agent or in the enzyme composition. Certain active ingredients, such as a substrate for the enzyme of interest, preferably are present in the composition comprising the film-forming agent.
Specific cosmetic preparations further include the usual components.
The composition comprises a vehicle to enable the active ingredient to be conveyed to the skin. Vehicles include water, solids and liquids. These are classified as emollients, emulsifiers, surfactants, solubilizers, propellants, solvents, humectants, thickeners and powders. The composition further may comprise a preservative to inhibit microbial outgrowth.
Emollients include alkyl higher fatty acids, natural oils, higher fatty alcohols, glyceryl and isopropyl esters, mineral oils, fatty alcohol esters.
Emulsifiers comprise compounds having a HLB (hydrophilic/lipophilic balance) value which is in the lower or in the higher ranges, i.e. compounds which are able to form a water-in-oil or compounds which are able to form an oil-in-water emulsion, respectively.
Thickeners include polysaccharides, gums and carboxylic group-containing polymers
Powders include chalk, talc, starch.
The method of the invention is suitable for any form of topical application, in the cosmetic as well as the dermatological field, wherein the action of an enzyme is required. The method of the invention is particularly suitable for topical application of a protease. Proteases are typically applied in cosmetic compositions for their cleansing, skin smoothening, anti-hairgrowth and anti-acne properties.